Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin.

Chun J; Auer KA; Jacobson BS

摘要

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKC epsilon, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading.

机译：海拉细胞在凝胶基质扩散需要激活的蛋白激酶C(PKC)发生的结果cell-attachment-induced激活磷脂酶A2 (PLA2)生产花生四烯酸酸(AA)和新陈代谢lipoxyginase的AA(LOX)。影响肌动蛋白,激活microtubule-based细胞骨架机械促进海拉细胞在凝胶扩散。明胶是取决于PKC蔓延诱导肌动蛋白聚合和microtubule-facilitated胞外分泌,根据以下的观察。丝状的相对含量增加(F)肌动蛋白在海拉细胞传播,海拉细胞治疗与PKC-activating佛波醇酯等12-O-tetradecanoyl佛波醇13-acetate (TPA)进一步增加相对内容的f -肌动蛋白和速度和程度细胞的扩散。PKC通过calphostin C阻止细胞扩散和f -肌动蛋白的增加内容。增加f -肌动蛋白PKC诱导的内容在悬浮细胞活化剂也被观察到与TPA治疗,f -肌动蛋白的动力学类似的PKC激活。此外,PKCε,PKC同种型大多数参与调节海拉细胞扩散为了应对生产AA,更迅速转移到膜对TPA的回应治疗比f -肌动蛋白的增加。阻塞PLA2或液态氧的活动抑制f -肌动蛋白形成和细胞传播,这两个被TPA治疗逆转。这个结果符合AA和液态氧代谢物AA是上游PKC激活及其的使者f -肌动蛋白形成和调节细胞蔓延。聚合在整个身体的细胞不仅在细胞基质粘附的地区不仅因为PKC激活有关与基底外侧质膜域联系的培养皿也顶端暴露在等离子体膜域培养基和细胞内的膜分数。f -肌动蛋白形成,激活PKC诱导的exocytotic upregulationβ1整合素细胞表面的胞内域,可能microtubule-dependent的方式因为upregulation抑制诺考达唑。生产及其cell-attachment-induced AA新陈代谢的液态氧活化的结果PKC,调控的双重角色在海拉细胞细胞骨架机械蔓延。f -肌动蛋白诱发的结构重组细胞传播,,另一个是exocytotic upregulation胶原蛋白受体细胞表面增强细胞扩散。

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin.

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